Subunit D of RNA Polymerase from Methanosarcina acetivorans Contains Two Oxygen-labile [4Fe-4S] Clusters

The [4Fe‐4S] clusters of Rpo3 are key determinants in the post Rpo3/Rpo11 heterodimer formation of RNA polymerase in Methanosarcina acetivorans

“…First, to determine if each variant Rpo3 was capable of forming a heterodimer with Rpo11, each variant was coexpressed along with histidine‐tagged Rpo11 [Rpo11(His)] in E. coli followed by purification of Rpo11(His) using Ni 2+ affinity and size‐exclusion chromatography. Similar to previous results obtained for the purification of recombinant Rpo3/11(His) and Rpo3ΔD3/11(His) heterodimers (Lessner et al., ), each single cluster‐binding Rpo3 variant was capable of forming a recombinant heterodimer with Rpo11(His) that was devoid of Fe‐S clusters (Fig. S1).…”

“…Previously, expression of Rpo3 deleted of D3/FLD, named Rpo3ΔD3 (previously DΔD3), in E. coli and within M. acetivorans , revealed that the FLD is not required for Rpo3 to form a heterodimer with Rpo11 (Lessner et al., ). To ascertain the importance of each [4Fe‐4S] cluster to Rpo3/11 heterodimer formation and the interaction of the Rpo3/11 heterodimer with other RNAP subunits during assembly of RNAP, Rpo3 variants were generated defective in binding cluster 1 or cluster 2.…”

“…The remaining sequences were aligned using the MEGA program (v 6.06) (Tamura, Stecher, Peterson, Filipski, & Kumar, ) to identify those sequences that contain a domain similar to domain 3, defined as aligning with residues 171–221 of the M. acetivorans sequence. Species were classified into six groups based on the presence of D3/FLD and arrangement of the cysteine residues comprising the predicted [4Fe‐4S] cluster‐binding motifs within the domain as described previously (Lessner et al., ).…”

“…The iron and acid‐labile sulfide content of Rpo3/11(His) heterodimers were determined as described previously (Cruz & Ferry, ). SDS‐PAGE and western blotting were performed by standard procedures, using anti‐Rpo3/11 antibody described previously (Lessner et al., ). Antibodies used to specifically detect Rpo3, subunit B′, or subunit A″ were supplied by Genscript and produced using synthesized peptides specific for each protein: Rpo3 (CISSDPKIQPADPNV), Rpo2′ (CGKTSPPRFLEEPSD), and Rpo1″ (CDGEVKQIGRHGISG).…”

Manipulating Archaeal Systems to Permit Analyses of Transcription Elongation-Termination Decisions In Vitro

“…Many archaeal RNAPs contain Fe-S centers [46, 47], however, there is no evidence for such clusters in T. kodakarensis RNAP and the enzyme can be purified aerobically without concern. Strains wherein a gene encoding a single subunit of RNA polymerase is modified to encode a protein with a His 6 -tag are now routinely used for purification of RNAP [39].…”